Angiogenesis means the formation of new blood vessels from pre-existing vessels by the growth, differentiation and migration of endothelial cells and does not occur in healthy adults, except for some special occasions, including wound healing, menstruation, etc. However, the excessive formation of new blood vessels in diseases, such as tumor growth and metastasis, age-related macular degeneration, rheumatoid arthritis, diabetic retinopathy, psoriasis and chronic inflammation, has been reported (Cameliet and Jain, Nature, 407:249, 2000). For this reason, many efforts to treat diseases, particularly tumors, using angiogenesis inhibitors, have been made.
Factors involved in angiogenesis include vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-b (TGFb), fibroblast growth factor (FGF), etc. Among them, the vascular endothelial growth factor is an endothelial cell-specific factor which is involved directly in the growth, differentiation and migration of endothelial cells, and there are four different isoforms (VEGF165, VEGF121, VEGF189 and VEGF206). Among the four isoforms, VEGF 165 is the most abundant isoform in all human tissues except placenta (Tisher et al., J. Biol. Chem., 266:11947, 1991).
Vascular endothelial growth factor (VEGF) regulates new blood vessel formation resulting from the differentiation of endothelial precursors (angioblasts) in situ, is expressed in embryonic tissues (Breier et al., Development (Camb), 114:521, 1992), macrophages, and proliferating epithelial keratinocytes during wound healing (Brown et al., J. Exp. Med., 176:1375, 1992), and may be responsible for tissue edema associated with inflammation (Ferrara et al., Endocr. Rev., 13:18, 1992). In situ hybridization studies have demonstrated high VEGF expression in a number of human tumor lines including glioblastoma multiforme, hemangioblastoma, central nervous system neoplasms and AlDS-associated Kaposi's sarcoma (Plate et al., Nature, 359:845, 1992; Plate et al., Cancer Res., 53: 5822, 1993; Berkman et al., J. Clin. Invest., 91:153, 1993; Nakamura et al., AIDS Weekly, 13(1), 1992). High levels of VEGF were also observed in hypoxia induced angiogenesis (Shweiki et al., Nature, 359:843, 1992).
The biological function of VEGF is mediated through its high affinity VEGF receptors which are selectively expressed in endothelial cells during embryogenesis (Millauer et al., Cell, 72:835, 1993) and during tumor formation. VEGF receptors (VEGFR) typically are class III receptor-type tyrosine kinases characterized by having several, typically 5 or 7, immunoglobulin-like loops in their amino-terminal extracellular ligand-binding domain of a receptor (Kaipainen et al., J. Exp. Med., 178:2027, 1993). The other two regions include a transmembrane region and a carboxy-terminal intracellular catalytic domain interrupted by an insertion of hydrophilic interkinase sequences of variable lengths, called the kinase insert domain (Terman et al., Oncogene, 6:1677, 1991). VEGF receptors include fms-like tyrosine kinase receptor (Flt-1), or VEGFR-1 (Shibuya et al., Oncogene, 5:519, 1990; WO 92/14248; Terman et al., Oncogene, 6:1677, 1991), kinase insert domain-containing receptor/fetal liver kinase (KDR/Flk-1), or VEGFR-2 (Matthews et al., PNAS, 88:9026, 1991), although other receptors such as neuropilin-1 and neuropilin-2 can also bind VEGF. Another tyrosine kinase receptor, VEGFR-3 (Flt-4), binds the VEGF homologues VEGF-C and VEGF-D and is important in the development of lymphatic vessels.
High levels of Flk-1 are expressed by endothelial cells that infiltrate gliomas (Plate et al., Nature, 359:845, 1992). Flk-1 levels are specifically upregulated by VEGF produced by human glioblastomas (Plate et al., Cancer Res., 53:5822, 1993).
The finding of high levels of Flk-1 expression in glioblastoma associated endothelial cells (GAEC) indicates that receptor activity is probably induced during tumor formation since Flk-1 transcripts are barely detectable in normal brain endothelial cells. This upregulation is confined to the vascular endothelial cells in close proximity to the tumor. Blocking VEGF activity with neutralizing anti-VEGF monoclonal antibodies (mAbs) resulted in inhibition of the growth of human tumor xenografts in nude mice (Kim, K. et al., Nature, 362:841-844, 1993), indicating a direct role for VEGF in tumor-related angiogenesis.
Although VEGF ligands are upregulated in tumor cells, and the receptors thereof are upregulated in tumor infiltrated vascular endothelial cells, the expression levels of VEGF ligands and the receptors thereof are low in normal cells that are not associated with angiogenesis. Therefore, such normal cells would block the interaction between VEGF and the receptors thereof to inhibit angiogenesis, thus inhibiting tumor growth.
High levels of VEGFR-2 are expressed by endothelial cells that infiltrate gliomas, and are specifically upregulated by VEGF produced by human glioblastomas (Plate et al., Nature, 359:845, 1992; Plate et al., Cancer Res., 53:5822, 1993). The finding of high levels of VEGR-2 expression in glioblastoma associated endothelial cells (GAEC) suggests that receptor activity is induced during tumor formation, since VEGFR-2 transcripts are barely detectable in normal brain endothelial cells.
Therefore, studies focused on inhibiting the activity of VEGF, which is expressed in tumor growth sites, to inhibit angiogenesis so as to inhibit tumor growth, are being actively conducted. Typically, methods of inhibiting VEGF receptors on the membrane of cancer cells to prevent VEGF from entering cells have been developed. Examples of cell lines producing VEGFR antibodies include a hybridoma cell line producing rat anti-mouse VEGFR-2 monoclonal antibody (DC101; ATCC HB 11534), a hybridoma cell line (M25, 18A1; ATCC HB 12152) producing mouse anti-mouse VEGFR-2 monoclonal antibody mAb 25, and a hybridoma cell line producing mouse anti-mouse VEGFR-2 monoclonal antibody mAb 73 [(M73,24; ATCC HB 12153), KM1730(FERM BP-5697; WO 98/22616; WO 99/59636), KM1731 (FERM BP-5718), KM1732 (FERM BP-5698), KM1748 (FERM BP-5699), KM1750 (FERM BP-5700)].
There has been a continuous development of humanized antibodies against VEGF receptors. These humanized antibodies against VEGF receptors, developed to date, showed high competition with VEGF in vitro, but had problems in that their ability to neutralize VEGF receptors in cells is reduced and in that the antibodies do not show cross-reactivity in mice or rats, such that animal tests cannot be carried out.
Accordingly, the present inventors have constructed a library of non-immunized fully human antibodies, screened single chain variable fragment (ScFv) antibodies against VEGF receptor (KDR), and found that the antibodies exhibit an excellent KDR-neutralizing effect not only in vitro, but also in cells and in vivo, and show cross-reactivity even in mice and rats, thereby completing the present invention.